1. Field of the Invention
This invention relates to rapid protection against alphavirus-induced encephalitides before or after exposure to alphaviruses. The protection can be achieved by the administration of a single dose, fast-acting vaccine comprising recombinant viral vectors expressing the structural proteins of alphaviruses or by the administration of a single dose recombinant viral vectors expressing interferon. Alternatively, the rapid pre- or post-exposure protection can be achieved by a single dose, simultaneous administration of the vaccine and the interferon delivered by the recombinant viral vectors or by a single dose administration of recombinant viral vectors co-expressing both the structural proteins of alphaviruses and interferon.
2. Brief Description of the Related Art
Alphavirus-induced encephalitides are caused by Venezuelan (VEEV), eastern (EEEV) and western (WEEV) equine encephalitis viruses (Griffin, 2001). The disease is characterized by fever, persistent headache, confusion, agitation, difficulty waling, and seizures. In severe cases, persistent neurological damage and death may occur. VEEV, EEEV, and WEEV cause encephalitides in humans with different fatality rates. EEEV is the most virulent with case fatality rates of 30% to 40%. In fatal cases, patients usually die within 2 to 10 days after the onset of encephalitis. Compared to EEEV, WEEV appears to be less virulent with case fatality rates of 10%. The fatal encephalitis often occurs in infants and in young children. VEEV usually causes an acute incapacitating illness with fever, chills, headache, muscle pain, diarrhea and vomiting. The encephalitis occurs usually in children with a case fatality rate of less than 1%. Besides fatal encephalitides in humans, VEEV, EEEV and WEEV also cause outbreaks of the disease in equines and other domestic animals with high death rates. Therefore, these viruses are important emerging and reemerging human and veterinary pathogens.
The structure of alphaviruses consists of a protein coat termed envelope, a capsid, and inside the capsid, a positive-sense, single-stranded RNA genome (Schlesinger and Schlesinger, 2001). The 5′ two thirds of the viral RNA genome encodes the nonstructural proteins required for transcription and replication of the viruses. The 3′ one third of the viral genome encodes the capsid and envelope proteins. The envelope proteins are encoded by a subgenomic mRNA and derived by the proteolytic cleavage of the E3-E2-6K-E1 polypeptide into the E2 and E1 proteins (Strauss and Strauss, 1994). E2 protein binds to E1 to form the spikes on the surface of the virion. The E2 and E1 proteins of alphaviruses trigger host immune responses against the viruses (Das et al., 2004; Hodgson, Ludwig, and Smith, 1999; Mathews and Roehrig, 1982).
VEEV, EEEV, and WEEV are transmitted by mosquitoes. Natural outbreaks of the disease occur when humans or domestic animals are bitten by mosquitoes carrying the viruses. There are no human-to-human transmissions of the viruses. Therefore, humans are considered to be dead-end hosts. Outbreaks of alphavirus-induced encephalitides can have enormous impact on human health. For instance, an outbreak of VEEV in Venezuelan and Colombia in 1995 caused more than 75,000 cases with 300 deaths reported (Weaver et al., 1996). About 1,000 human cases were recorded in 1941 in a major outbreak of WEEV occurred in western Canada (Reisen and Monath, 1989).
Besides natural outbreaks, VEEV, EEEV, and WEEV are potential bioterrorism and biowarfare agents because they are highly infectious through the aerosol transmission. For example, only 10 to 100 of the aerosolized VEEV are sufficient for infection and at least 150 human cases of laboratory acquired infections have been reported (Sidwell and Smee, 2003). The U.S. Centers for Disease Control and Prevention (CDC) has classified VEEV, EEEV, and WEEV as category “B” bioterrorism agents.
Currently, there were no vaccines, antiviral drugs or therapeutics for rapid pre-exposure prevention or post-exposure protection against alphavirus-induced encephalitides. For pre-exposure prevention, a live attenuated VEEV vaccine, designated as TC-83, has been developed. The vaccine protects laboratory workers from the VEEV infection. However, 15% to 30% of vaccine recipients developed fever, malaise and headache and these side-effects are so severe that bed rest was required in about the half of vaccine recipients (Hoke, 2005). Killed EEEV and WEEV vaccines with Investigational New Drug (IND) status are available only for laboratory workers at the risk of exposure to these viruses. However, to be effective, these EEEV and WEEV vaccines require multiple injections and annual booster. Therefore, improved vaccines are needed for the rapid pre-exposure prevention of alphavirus-induced encephalitides.
Because no antiviral drugs or therapeutics are available for post-exposure protection against alphavirus-induced encephalitides, the treatment focuses on easing clinical symptoms using antihyperthermia procedures for fever and anticonvulsant drugs.
Several strategies have been proposed for post-exposure protection against alphavirus-induced encephalitides. The first is the use of mouse monoclonal antibodies (mAbs) that neutralize alphaviruses. Such mAbs protected approximately 50% mice against VEEV infection when given 24 h after the airborne challenge with the virulent VEEV (Phillpotts, 2006; Phillpotts, Jones, and Howard, 2002). Because mouse mAbs are highly immunogenic in humans and are not suitable for human use, these mouse mAbs are humanized by replacing much of amino acid sequence of mouse mAbs with those of humans. Ninety percent of the VEEV infected mice were cured after they were given the humanized mAbs within 1 h after exposure to VEEV and 75% of mice were cured after they were given the humanized mAbs 24 h after the virus exposure (Hunt et al., 2006). However, a drawback of using mAbs for post-exposure protection against alphavirus-induced encephalitides is that a large quantity of purified mAbs is required. For instance, as much as 4 mg/kg mAbs is needed to protect 50% of mice from VEEV infection (Phillpotts, Jones, and Howard, 2002). Although mAbs against WEEV have been developed (U.S. Pat. No. 6,812,329) (Long et al., 2000a; Long et al., 2000b; Yamamoto et al., 1985), there have been no reports regarding their protection of animals against WEEV infection.
Another strategy for post-exposure protection against alphavirus-induced encephalitides is the use of interferon alpha (IFN-α). IFN-α is produced by cells immediately after virus infection. It inhibits the replication of a wide-spectrum of viruses by inducing cells to synthesize various antiviral proteins. U.S. patent application Ser. No. 11/231,433 (Publication Number US2006/0024270) discloses methods for treating patients with viral encephalitides by injection of IFN-α. Compared to patients that did not receive IFN-α, patients given IFN-α resulted in a significantly improved neurologic function and survival. However, clinical use of IFN-α for viral encephalitides is limited by the requirement of frequent injections (typically every 24 h for up to 14 days) with a large dose of IFN-α (3 million units) and toxicities associated with IFN-α, which include a flu-like syndrome with fever, malaise and headache. Conjugating IFN-α with polyethylene glycol (PEG) prolongs the in vivo half-life of IFN-α and its potency. For instant, pre-exposure treatment of mice with PEG-conjugated IFN-α prevented mice from either a subcutaneous or an aerosol challenge of VEEV while pre-exposure treatment of unmodified IFN-α did not (Grieder and Vogel, 1999; Lukaszewski and Brooks, 2000). However, there are no reports to define if PEG-conjugated IFN-α could protect mice from encephalitides after the exposure to VEEV, WEEV or EEEV.
Several experimental vaccines for VEEV, EEEV and WEEV have been developed for the prevention of alphavirus-induced encephalitides. However, no data have been shown that these experimental vaccines are effective for post-exposure protection. U.S. Pat. No. 6,261,570 describes the construction of live attenuated vaccines for these alphaviruses. A single dose injection of these vaccines protected animals from the challenge of the viruses. However, concerns of side effects and the reversion to the virulence wild-type viruses may prevent their use in humans. A DNA vaccine candidate for WEEV completely protected mice from the challenge of WEEV (Nagata et al., 2005). Similar to the killed WEEV vaccine, however, the DNA vaccine candidate requires three injections to be effective. Several groups demonstrated that viral-vectored VEEV vaccines protected mice from VEEV challenge (Paessler et al., 2006; Perkins, O'Brien, and Phillpotts, 2006; Phillpotts et al., 2005; U.S. Pat. No. 6,936,257). U.S. Pat. No. 6,565,853 teaches an adenovirus which encodes a polypeptide which produces a protective immune response against an alphavirus, such as a VEEV, when administered to a mammal.
Finally, several anti-alphavirus drug candidates have been developed. For instance, Poly ICLC, a nucleic acid-based immunomodulator, induces interferon production and activates natural killer cells. Liposomes encapsulated poly ICLC offers 100% protection against a lethal intranasal challenge of WEEV (Wong et al., 2005). However, no data have been reported on post-exposure protection. Other drug candidates such as triaryl pyrazolin (Puig-Basagoiti et al., 2006), ribozyme (Seyhan et al., 2002), the zinc-finger antiviral protein (Bick et al., 2003), and human lactoferrin (Waarts et al., 2005) inhibit alphavirus replication in a cell culture system; however, it is unknown if these drug candidates are effective in the prevention or treatment of alphavirus-induced encephalitides in animals.
Thus, this invention addresses two important aspects of medical countermeasures against alphaviruses: one is to rapidly prevent alphavirus-induced encephalitides before exposure to the viruses and another is to rapidly protect against alphavirus-induced encephalitides after exposure to the viruses. The present invention can be used to prevent the encephalitides should the following scenarios occur: a bioterrorism attack or a biowarfare in which the viruses are deliberately released to the public or to military personnel, a natural outbreak of VEEV, or EEEV or WEEV, and a laboratory accident in which the laboratory workers are at the risk of exposure to these viruses.